The adaptive immune system encodes the ability to remember an initial encounter with an antigen and to respond to that antigen upon re-exposure in a rapid and robust fashion for the life time of the individual. This phenomenon of immunological memory is a fundamental property of the adaptive immune system and is the basis for all vaccine development. For most vaccines, neutralizing antibodies plays a critical role in protective immune responses, and thus the mechanisms that underlie the generation and maintenance of humoral memory are of considerable interest. Long-term humoral immunity is encoded in memory B cells (MBC) and long-lived plasma cells (LLPC) which are generated as a part of the primary immune response. LLPC are terminally differentiated cells that reside in the bone marrow and are responsible for the long-term maintenance of serum Ab levels which play a key role in the initial control of pathogens and their toxins upon reinfection. MBC are capable of mounting an antigen-induced response by proliferating and differentiating into plasma cells (PC) resulting in rapid, high titer secondary Ab responses upon re-exposure to pathogens. Despite the central role of MBC in combating infections, our understanding of the cellular and molecular mechanisms that underlie the generation, maintenance and reactivation of B cell memory is incomplete. Efforts to develop new vaccines would benefit from a more detailed knowledge of these processes, particularly vaccines against a pathogen such as Plasmodium falciparum in malaria which appear to subvert immunological memory. This project represented a collaborative effort between Dr. Pierce and Dr. Louis Miller and his colleagues in the Malaria Vaccine Development Branch (MVDB). Over the last year we have focused our efforts on gaining an understanding of the generation, maintenance and activation of B cell memory in naive individuals in response to vaccination. Of particular interest in this process is the role of TLR9, a pattern recognition receptor that initiates innate immune responses. TLR9 detects microbial DNA with hypomethylated CpG motifs and in humans is preferentially expressed by plasmacytoid dendritic cells (PDC) and B cells. TLR9 ligands have been indirectly implicated in the maintenance of B cell memory although at present the role of TLR9 in the generation of B cell memory has not been addressed. We have taken advantage of recent advances in the identification of antigen-specific human MBCs in peripheral blood to describe the generation and maintenance of malaria-specific MBCs in the U.S. in response to vaccines currently under development in the MVDB. In collaboration with the MVDB, we described the acquisition of antigen-specific MBCs in the peripheral blood of volunteers enrolled in two trials of the malaria vaccines, one composed of P. falciparum apical membrane antigen 1 (AMA1) and one of merozoite surface protein 1 (MSP1), both on alum either alone or in combination with the TLR9 agonist, CpG. We found that the acquisition of MBCs is a dynamic process in which the antigen-specific MBC pool rapidly expands and then contracts following vaccination. In individuals who received CpG-containing vaccines, antigen-specific MBCs appeared more rapidly, in greater numbers, and persisted for longer. The percentage of vaccine-specific MBCs present at the time of re-immunization predicted antigen-specific antibody levels 14 days later and at steady state, there was a positive correlation between antigen-specific MBCs and antibody levels. We also observed an antigen-independent decrease in the total IgG+ MBC pool in circulation 3 days after each vaccination, possibly the result of adjuvant-induced trafficking of MBCs into tissues. Consistent with this possibility we observed a large increase in the total number of PCs in circulation following vaccination, suggesting that MBCs induced to leave the circulation gave rise to plasma cells. These are the first data describing the naive human MBC response to vaccination. We recently completed a similar analysis in semi-immune adults living in a malaria-endemic area of Mali. In contrast to our observations in nonimmune U.S. volunteers, we found that although vaccination with AMA1 induced a transient increase in the frequency of AMA-1 specific MBCs, the inclusion of CpG in the vaccine had no measurable effect on the response. In Malian adults we also failed to see a correlation between AMA-1 specific MBC frequencies and the levels of AMA-1 specific antibody. Thus, adults living in malaria endemic areas appear to be relatively refractory to the potential effects of CpG-TLR9 activation. Studies are in progress to understand the cellular and molecular basis of the relative refractoriness to CpG in Malian adults. We recently expanded the scope of our MBC studies to address the molecular basis of the enhanced responses of MBCs. Using live cell total internal reflection fluorescence (TIRF) microscopy we are analyzing the behavior of the BCR expressed by human CD19+CD27+IgG+ MBCs as the B cells first encounter antigen. We will also determine the dependence of MBC activation on the B cell adaptor CD19 and its sensitivity to the effects of the B cell inhibitory receptor, FcgammaRIIB. Through these studies we hope to gain knowledge of the initiation of MBC response that may guide vaccine design.